Cellular and molecular mechanisms involved in T cell-mediated autoimmunity against immunologically privileged retinal antigens are being studied. The questions are aimed at elucidating the natural development and maintenance of self-tolerance to retinal antigens, and at defining the processes that lead to their pathological breakdown. The goal is to use this knowledge for designing novel and rational strategies for immunotherapy. The experimental approaches utilize the model of experimental autoimmune uveoretinitis (EAU), which resembles immune-mediated uveitic diseases in humans that can lead to blindness. EAU is induced in mice and rats by immunization with retinal antigens such as IRBP, Arrestin, or their component peptide epitopes, or by infusion of cultured lymphocytes that recognize these antigens. The mechanisms controlling disease susceptibility and pathogenesis are being defined at the genetic, developmental, and immunological levels. Novel approaches to disease regulation are devised based on these findings. Some recent findings are described ahead. The nature and extent of tolerance to immunologically privileged self antigens is poorly understood. Development of tolerance to self during ontogeny is being investigated by using mice that have differential expression of IRBP, all bred onto the EAU-susceptible B10.RIII background. 1) IRBP-transgenic (TG) mice that express the N-terminal half of IRBP extraocularly under control of the MHC class II promoter. 2) IRBP knockout (KO) mice that have no detectable expression of IRBP and 3) wild type (WT) mice expressing IRBP in the eye and pineal. We show that extraocular expression of a privileged retinal antigen in IRBP TG mice abolishes the ability to develop EAU in a transgene-specific fashion. Studies with IRBP KO mice indicate that thymic selection plays a major role in tuning the threshold of susceptibility to ocular autoimmunity. Expression of IRBP in the thymus by immunostaining was detectable in WT and TG, but not in KO mice. In keeping with this, IRBP-KO mice have strongly enhanced immunological responses across the board to IRBP and show a changed recognition of IRBP epitopes, suggesting an expanded T cell repertoire that had not undergone negative selection. Thymic transplantation between IRBP KO and WT show this thymic expression to be functionally relevant and to change susceptibility to EAU development. All immunological responses of thymectomized recipients implanted with a mismatched thymus resembled the pattern characteristic of the thymus donor. The contribution of peripheral mechanisms to tolerance is being investigated. These studies will help understand how the IRBP specific T cell repertoire is generated and controlled. As a complementary approach, mice transgenic for a uveitogenic T cell receptor are in the process of being developed. These mice will provide a powerful tool to study the selection, differentiation and trafficking of antigen-specific T cells in this model. EAU, similarly to uveitis in humans, is genetically controlled by MHC as well as non-MHC genes. We study genetic control of EAU using two approaches: 1) defining genetic markers associated with susceptibility in genetically defined rodents. This approach previously identified 3 chromosomal regions affecting EAU susceptibility in rats, which co-localize with a number of immunologically relevant loci. Consomic rat strains are now being bred by marker-assisted selection, in which each of these chromosomes from the susceptible background is transferred to a resistant background, and vice versa. Genes differentially expressed by susceptible and resistant strains are being identified using DNA microarrays. These studies will permit closer identification of the genes and pathways determining susceptibility or resistance to EAU. 2) We have developed a ?humanized? model of EAU using HLA-transgenic mice in which murine MHC genes have been replaced by human MHC genes. The humanized EAU model demonstrates that presentation of retinal antigens by MHC molecules can lead to retinal disease and provide a tool to identify the epitopes involved. Interestingly, one of the ?humanized? strains is developing strong EAU when immunized with S-Ag, which induces little or no uveitis in WT mice. The HLA transgenic mice have an altered T cell repertoire as evaluated by epitope recognition, and respond to a major uveitogenic epitope of S-Ag that is recognized by uveitis patients. These results validate EAU as a model of human uveitis. Efforts have been made to delineate the importance of adjuvant effects in EAU. Bacterial toxins, such as cholera toxin (CT) and pertussis toxin (PT) are examples of bacterial products that trigger innate immune responses and act as immune adjuvants. These toxins are typically composed of an enzymatic A subunit, and of a cell binding B subunit that delivers the A subunit into the cell. PT has been used for years to promote cell mediated autoimmunity in animal models, but the mechanisms are poorly understood. In earlier studies, we found that PT given at the time of immunization promotes development a Th1 response to the retinal antigen, but if given later, at the time of effector cell migration to the target organ, it actually prevents disease by blockade of chemokine signaling through G-protein coupled receptors. We have now been able to resolve these opposing effects of PT, and show that whereas the disease-inhibitory ability resides in the A subunit, the disease-enhancing activity resides primarily in the B subunit. In contrast to PT, CT appears to inhibit EAU through induction of immune deviation. These studies help to understand disease modulation by adjuvant effects. Genetic approaches to EAU immunotherapy are being explored. We have generated a plasmid containing the first repeat of IRBP and have used it for DNA vaccination as a means to prevent or reverse EAU. Two vaccination protocols were used, IV and IP. In both cases the IRBP protein was shown to be expressed in tissues after plasmid administration, and both approaches showed efficacy in preventing EAU when given prior to immunization. The IV protocol was further explored as to mechanism. Data suggest that there is induction of immunological hyporesponsiveness, but without evidence for immune deviation or for regulatory cells. Bcl-2 regulated apoptosis was also eliminated as a mechanism. In a disease reversal protocol, where vaccinated animals were challenged with a uveitogenic effector T cell line there was a modest, but consistent benefit. This suggests that DNA vaccination targets primarily naive T cells, with a minor effect on effector/memory cells. A peptide-MHC-class II dimer that binds to the antigen receptor of T cells specific for the major IRBP epitope, peptide 161-180, was generated in collaboration with J. Schneck of Johns Hopkins U, by fusing the 161-180 sequence with I-Ar on an IgG scaffold. This reagent, expressed in baculovirus, will serve as a tool to detect and to modulate antigen-specific T cells and to by binding to their T cell receptor. This can serve as a diagnostic tool as well as another approach to antigen-specific therapy. Methimazole (MMI), a compound used clinically in therapy of Graves? thyroiditis, inhibited induction of the EAU and associated antigen-specific responses. Contrary to previous notions the in vivo protective effect was IFN-g-independent. The data suggested that MMI inhibits EAU at least in part by preventing the recruitment and/or maturation of APC, resulting in reduced generation of Ag specific T cells. Because this agent is already approved for use in the clinic, its use as a potential immunomodulator in uveitis is an attractive notion.